Laing, my supervisor, for his stimulating thoughts, sound advice and genuine support for my decisions throughout the project. John Bower for his advice and for access to his physiology laboratory and resources.
Introduction
The free-living heterotrophic aerobic bacteria fix nitrogen non-symbiotically or loosely in association with grasses (Krishnamoorthy and Rema, 2004). The aim of this study was to find nitrogen fixation conditions for free-living diazotrophs (FLD) in chemically defined, nitrogen-free media.
Biofertilizers
Vadakattu and Paterson (2006) explained that there is a wealth of free-living bacteria in soil that are capable of fixing significant amounts of atmospheric nitrogen in the absence of legumes, using crop residues and root exudates as energy sources. Azotobacter is one of the plant growth-promoting rhizobacteria (PGPR) that can benefit plants by colonizing their root zone (Ahmad et al., 2006).
Free-living nitrogen fixing bacteria
The Genus Azotobacter
The Genus Azospirillum
Isolation of nitrogen-fixing bacteria
The Genus Azotobacter
The Genus Azospirillum
Growth media
Factors affecting growth
- Temperature
- pH
- Inorganic salts
- Oxygen
A deficiency of Ca in the medium leads to an extension of the lag phase, but its action is not considered to be specific for nitrogen fixation. When Azotobacter grows in an N-free culture, increasing dissolved oxygen tension improves cell concentration (Pena et al., 2000).
Cyst formation
Addition of FeSO4 reduced the number of viable cells of a mutant strain of Azotobacter vinelandii Lipman (Edwards et al., 2000); while in another study it increased growth (Vermani et al., 1997). However, the concentration of Ca salts must not exceed a certain optimum (Mishustin and Shilnikova, 1969), because higher levels of Ca are inhibitory.
Shelf life
Cells accumulate poly-beta-hydroxybutyrate and grow into chains or filaments that eventually lose motility and form capsules.
Factors affecting nitrogen-fixation
- Temperature
- pH
- Oxygen
- Inorganic Salts
- Nitrogen
The nitrogenase complex requires magnesium ions to be active, so the magnesium requirement for nitrogen fixation is substantial (Sylvia et al., 1999). In many cases where nitrogen fixation is measured as nitrogenase activity, it is reduced in the presence of pooled nitrogen in the medium (Laane et al., 1980).
Detection of nitrogen-fixation
The stable isotope (N 15 ) method
Vanadium is known to stimulate nitrogen fixation in a number of organisms, including several species of Azotobacter, some cyanobacteria and phototrophic bacteria, and Clostridium pasteurianum Winogradsky (Brock et al., 1994). Detection of 15N in tissues or cells provides definitive evidence of nitrogen fixation and allows highly accurate quantification of the amount of nitrogen fixation (Sylvia et al., 1999).
The nitrogen difference method
After incubation, the samples are digested and the 15N content of the material is determined using a mass spectrometer. This method is accurate, but is time consuming and expensive, both in terms of equipment required and production of the 15N2.
Acetylene reduction assays (ARA)
Samples are exposed to an atmosphere of about 10% 15N2, usually in an argon or helium balance, to eliminate competition from 14N2.
Biofertilizer inoculants and nitrogen-fixation
Scope and potential application of FLD inoculants
FLD inoculants and their effects on various aspects of crop growth
Inoculants of Azotobacter spp
Furthermore, nitrogen concentration in wheat grains and roots can be increased due to Azotobacter bio-inoculants (Wadad and Vlassak, 1988; Kader et al., 2002). According to Oblisami et al. 1979), only the effect of Azotobacter inoculants resulted in better growth of root systems when compared to that due to growth regulators (gibberellins (GAs) and indole-3-acetic acid (IAA)) applied alone.
Inoculants of Azospirillum spp
ISN-129 increased seed yield and total dry matter, especially when no external nitrogen was applied (Singh and Bhargava, 1994). The increased nitrogen content was not the result of nitrogen fixation, but of the nitrate reductase activity of the bacteria in the roots.
Application in combination with other bio-inoculants, chemical fertilizers and
Effect of phosphate and oxygen concentration on alginate production and metabolic stoichiometry of Azotobacter vinelandii under micro-aerobic conditions. The influence of culture medium composition on the morphology and reproduction of Azotobacter chroococcum.
Introduction
Materials and methods
- Origin of soil samples
- Isolation, culture and media
- Screening of isolates
- Nitrogenase activity
- Statistical analysis
The aim of this study was to isolate a spectrum of FLD bacteria from a mixture of crop rhizospheres, to evaluate their growth in culture and their nitrogenase activity on nitrogen-free media. One colony of each isolate was placed on N-free Burke's medium (with mannitol as carbon source) and stirred at 150 rpm for 48 hours.
Results
- Isolation
- Growth of FLD in liquid enrichment cultures
- In vitro Screening of FLD Isolates on N-free media
- Microscopic observation on the morphological characteristics of FLD isolates
From enrichment cultures, FLDs were then purified onto similar solid media and assayed for colony formation. FLDs isolated from nitrogen-free media were assayed for their nitrogenase activity using the method described by Turner and Gibson (1980).
Means followed by the same letter are not significantly different, using Duncan’s
Growth of FLD on different media, temperature and pH
Isolates L1 and G3 grew well on the ethanol medium at all temperature and pH values (Table 2.2). This isolate also grew well on Jensen's Medium at 200C pH 7.0 and at 250C and all pH values, and slightly less vigorous growth on Burke's Medium at 200C and pH 7.0 and 8.0. On the other hand, isolate E9 grew poorly on the mannitol medium at all temperatures and pH evaluated. Isolate B3 grew well on Burke's and Jensen's media at all temperatures and pH 6.0 and 7.0 (Table 2.2). The best growth was shown by Isolate G3 and L1 on ethanol medium at all temperature and pH ranges (Table 2.2).
Means followed by the same letter are not significantly different using Duncan's New Multiple Test at alpha 0.05.
Discussion
Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects.
Introduction
Currently, strains belonging to the species Azotobacter vinelandii Lipman and Azotobacter chroococcum Beijerinck are used as soil inoculants in rainy areas and in warm and alkaline soils (Pandey et al., 1998). Burkholderia species are known to exhibit activities involved in nitrogen fixation, bioremediation, plant growth promotion or biological control in vitro (Caballero-Mellado et al., 2007). The members of the genus have been developed as crop inoculants for cereals (Wani et al., 1988), oilseeds, cotton and vegetables due to their ability to produce plant growth regulators (Dobbelaere et al., 2003), siderophores and to produce plant growth regulators (Dobbelaere et al., 2003), siderophores and ammonia (Garg et al. found that tomato growth increased when the crop was inoculated with Azotobacter.
Sixty to 80% of the nitrogen content of sugarcane is derived from BNF and it is believed that Acetobacter diazotrophicus Gillis et al.
Materials and methods
- Source of free-living diazotrophs
- Preparation of cell suspensions
- Crops evaluated
- Application methods
- Nitrogen analysis
- Statistical analysis
A prepared cell suspension (108 cells ml-1) of each free-living diazotrophic isolate was added separately to each of five Erlenmeyer flasks and labeled. Twenty milliliters of the cell suspension of the FLD isolate was added to each flask that already contained a 20 mL aliquot of the label. Appropriate numbers of maize seeds were placed separately in each of the five bacterial suspensions and mixed.
For elution, the cell suspension of FLD isolates was counted using a hemocytometer and adjusted to 108 cells ml-1.
Results
- FLD isolates applied as drenches to maize seedlings
- FLD isolates applied as seed treatments to maize crop
- FLD applied as drenches to sorghum seedlings
- FLD isolates applied as seed treatments to sorghum
- FLD isolates applied as drenches to wheat plants
- FLD isolates applied as seed treatments to wheat plants
- FLD isolates applied as drenches to lettuce seedlings
- FLD isolates applied as seed treatments to lettuce crops
- FLD applied as drenches to zucchini plants
- FLD isolates applied as seed treatments to zucchini plants
- FLD isolates applied as drenches to petunia seedlings
Significant differences (P<0.001) in wet weight, dry weight and yield were observed when FLD isolates were applied as seed treatment. Sorghum seedlings treated with a cell suspension of FLD isolates showed an increase in wet and dry biomass compared to the untreated control (P < 0.001). Plants grown from sorghum seeds treated with FLD isolates developed significantly greater wet and dry weight than the untreated control (P<0.001) (Table 3.13).
Isolate L1 was significantly more effective in increasing plant N compared to the untreated control and other FLD isolates (P<0.001) (Table 3.8 and Table 3.13).
Discussion
The FLD isolates were applied by drenching or seed treatment and in many cases significantly increased the growth and yield of corn, sorghum and wheat crops in greenhouse trials. Although there was no significant increase in plant N, FLD isolates applied by watering also improved the growth and fruit yield of zucchini plants. This could be due to the production of plant growth hormones and other biologically active substances by the FLD isolates (Rózycki et al., 1999).
The results showed that FLD isolates have the potential to fix nitrogen and promote the growth of various crops.
Biological nitrogen fixation (BNF), associated with cereals and grasses, contributes to the growth and nutrition of several crops. The results of our studies on the effects of FLD on growth and nitrogen fixation are consistent with the results of Boddey and Döbereiner (1988), who reported a significant increase in growth and nitrogen levels in forage grasses and sugarcane when inoculated with Azospirillum spp. Drip application of FLD isolates induced a growth-promoting effect on lettuce and petunia seedlings and improved plant-by-plant N in a greenhouse study.
The application of FLD to cereals (maize, sorghum or wheat) either drench or seed treatment application contributed to an increase in growth and plant N.
- Introduction
- Materials and methods
- Preparation of inoculants
- Microbial inoculation
- Controls
- Nitrogen total analysis
- Statistical analysis
- Results
- Effects of two FLD bacteria on wet and dry biomass and nitrogen
- Discussion
- References
Plant growth promoting rhizobacteria (PGPR) are commonly used as inoculants to improve the growth and yield of agricultural crops (Khalid et al., 2004). According to Bai et al. 2002), co-inoculation of plant growth promoting rhizobacteria (PGPR) strains increased nodule number, plant dry weight and fixed nitrogen at optimal dose (108 cells per seedling). Evaluation of genetic diversity and plant growth-promoting activities of nitrogen-fixing bacilli isolated from rice fields in southern Brazil.
Plant growth promotion by plant growth promoting rhizobacteria under greenhouse and two different field conditions.
- Introduction
- Materials and methods
- Production of the FLD isolate
- Source of Trichoderma
- In vitro dual culture of Isolate L1 (B. cereus) and T. harzianum
- Greenhouse trial
- Seedling drench
- Seeds
- Biomass measurements
- Nitrogen analysis
- Statistical analysis
- Results
- In vitro interaction between Eco-T ® (T. harzianum) and Isolate L1 (B. cereus)
- Discussion
- References
Mixed inoculations of isolate L1 (Bacillus cereus Frankland) at 106 and Eco-T® (Trichoderma harzianum Rifai) at 108 cfu ml - 1 resulted in a significant increase in growth and nitrogen content of lettuce. In order to determine the effects of the Eco-T® (T. harzianum) and Isolate L1 (B. cereus) treatments, we changed the plant growth parameters into percentages. Isolate L1 at a concentration of 108 cfu ml-1 alone and in combination with Eco-T® gave less wet and dry weight and plant N.
In the greenhouse experiment, either solo or double inoculation of FLD isolate L1 (B. cereus) with Eco-T® (T. harzianum) improved plant growth and nitrogen content of lettuce seedlings.
- Introduction
- Materials and methods
- Bacterial culture
- Hydroponics experiment
- Plant harvest, growth parameters and N-analysis
- Nitrogen analysis
- Statistical analysis
- Results
- Discussion
- References
Strains of Acetobacter and Herbaspirillum can contribute significant amounts of nitrogen to sugarcane (Kennedy et al., 1997). According to Maheshwari et al. 1992), use of FLD (mainly Azotobacter spp.) alone increased palmarosa (Cymbopogon martini var. Lettuce seedlings were used for evaluating the effect of FLD on plant growth and N levels due to the inoculation of microbial inoculant Isolate L1 (B . cereus) and different doses of a mineral fertilizer in hydroponics.
The response of lettuce growth and plant N to application of 25% of the recommended NPK level (25% NPK) integrated with Isolate L1 (B. cereus) was significantly better than that of the 25% NPK alone.
- Introduction
- Materials and methods
- Field experiment design
- Inoculum preparation
- Seed treatments
- Data collection
- Statistical analysis
- Results
- Estimation of FLD Populations
- Growth and yield parameters
- Discussion
- References
The smallest FLD population was in the rhizosphere of plants treated with NPK fertilizer, and plants of the untreated control. During the first month, FLD CFU counts in the rhizosphere of maize plants inoculated with isolate D6 were significantly higher than those of other treatments (Table 7.1). In the third month, the numbers of FLD bacteria were not significantly different between treatments, except for plants treated with isolate D6 (Table 7.1) and see (Fig. 7.2) in Appendix 7.2.
The number of FLD cells was significantly higher in the rhizosphere of plants that were inoculated with isolate D6, followed by Br2, L1 NPK and the untreated control.
- Introduction
- Conditions that affect growth of FLD and nitrogen fixing activity
- Current understanding
- Future research
- Application methods
- Current understanding
- Integrated treatments
- Integration of FLD with Eco-T ® (T. harzianum)
- Current understanding
- Future research
- Interaction of FLD with fertilization (NPK) in hydroponics
- Current understanding
- Future research
- References
One of the advantages of biological (microbial) nitrogen fixation is that it is largely regulated by the host plants, and nitrogen is supplied to host plants only on demand. Biological nitrogen fixation is sustainable in that energy sources are abundant from host plant exudates (a renewable source unlike non-renewable fossil fuels). Inoculation of FLD Isolate L1, combined with various doses of NPK fertilizer in a hydroponic system resulted in better nitrogen fixation at 25% NPK followed by 50%.
FLD growth and their nitrogen-fixing activities are affected by media, temperature, pH, and other conditions.
The same applies to Jensen's Medium, except that ethanol was used as the carbon source; 10 ml of ethanol was added to 1 liter of water from a filter-sterilized solution.
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